Field of the Invention
The present invention relates generally to the fields of detection and diagnosis and, more specifically, to diagnosis of the presence of early stage (primary) or late stage (metastatic) cancerous tumors, using a combinatorial approach of measuring circulating tumor stem cells and serum AnnexinA2.
Description of the Related Art
Developing relatively simple, non-invasive methods for diagnosing the presence of either benign tumors, such as colonic hyperplasia, adenomatous polyps, or cancerous tumors, such as adenocarcinomas at different stages of the disease, has remained a challenge. Early stages of epithelial cancers are defined as cancerous tumors limited within the tissues of origin, e.g., primary tumors, while later stages of cancer represent metastatic growths either within the lymph nodes or at distant sites away from the primary cancerous growth, such as metastatic growths in the lung/liver of colorectal carcinomas. With the advent of sophisticated proteomics in the past 7 years, investigators discovered that AnnexinA2 (ANXA2) is increasingly expressed by epithelial tumors in relation to stage of the disease (1-16). AnnexinA2 is normally present intracellularly and performs important functions of cellular trafficking (17). However, rapidly proliferating tumor cells (especially at leading edges of tumors) express membrane-associated extracellular, cell-surface associated AnnexinA2 (CS-ANXA2) (1-3,8,10-16,18). Other members of Annexin family (Annexin A4, Annexin A3) are reportedly over-expressed in ovarian cancers, and increase chemo resistance (19,20). Both tetrameric and monomeric forms of AnnexinA2 have been described on cell surface of endothelial and tumor cells (12,21).
As described above, surface-associated ANXA2 (CS-ANXA2) is increasingly expressed by many solid tumors, including colorectal (CRCs) and pancreatic cancers. ANXA2 lacks transmembrane domains and is tethered to the cell surface by binding to a 26Kda transmembrane protein (11,31). Surface-associated ANXA2 was significantly increased in colorectal adenomas and adenocarcinomas vs corresponding normal colonic mucosa (18).
Measuring circulating tumor cells in the blood of patients is a relatively new concept for diagnosing cancer, and is as yet in its infancy (52-55). Presence of circulating tumor cells likely predicts metastasis, and can be useful for monitoring recurrence (relapse) of the disease, post-treatment (56-60). Circulating tumor cells are detected by using many different methods, including immunocytochemistry (IHC) for epithelial markers such as cytokeratine-19 (CK19), and RT-PCR analysis for cancer-cell specific transcripts (61,62). Efficiency of such tests are poor, because of the rare presence of cancer cells within a very large number of blood cells (1-1000 CTCs in 109 blood cells/ml blood). Negative selection by excluding blood cells has been used to enumerate circulating tumor cells (63). Positive selection of circulating tumor cells using antibodies (Abs) against epithelial cell surface proteins such as cell adhesion protein EpCAM is currently being used (64). Circulating tumor cells, positive for EpCAM, are captured using microfluoridic CTC-Chip devices (64) and immunomagnetic bead-based methods (18,57). Isolated circulating tumor cells are confirmed by CK19 staining or Her2 amplification (53,65). Negative staining for CD45 (a Leukocyte marker) is also used (52). It is postulated that invasive cancer cells, going through epithelial mesenchymal transition (EMT), lose expression of cell surface EpCAM (66). Thus invasive circuiting tumor cells, with the highest metastatic potential, may be under-evaluated using anti-EpCAM Abs (52,66,67). Besides, efficacy of different EpCAM-Abs for capturing circulating tumor cells differs significantly (68). Thus, there is a recognized need in the art for efficient and effective methods of measuring circulating tumor cells.
The presence of CTCs in the blood will not detect the presence of benign tumors at a pre-cancerous stage, such as adenomatous polyps growing in the colon of the patients. The escape of cancer cells from a pre-cancerous growth, such as adenomas, is much less likely since the basement membrane for such tumors is still intact. However, the pre-cancerous benign tumorous growths are well vascularized with blood vessels, and tumor-specific antigens can be secreted into the blood supply. Significant levels of AnnexinA2 are present in the serum of patients with breast (2,3), hepatocellular (17), and lung (79) cancers. So while one can potentially diagnose and predict the presence of cancerous tumors using the newly described assays for either circulating tumor cells or cancer specific antigens, such as Her/neu, PSA or AnnexinA2, no diagnostic tests have been described to predict the presence of pre-cancerous, benign tumors (such as colon polyps) in the patients.
There is thus a recognized need in the art for effective methods of detecting the presence of benign pre-cancerous tumors, before they convert into cancerous growths, as a preventative measure. The present invention fulfills this long-standing need and desire in the art.